This application claims priority under 35 USC xc2xa7119 to Japanese Patent Application No. 2000-333363, filed Oct. 31, 2000, hereby incorporated by reference in its entirety.
The present invention relates to a novel nicotinamide adenine dinucleotide-dependent (R)-2,3-butanediol dehydrogenase. The present invention also relates to a polynucleotide encoding the enzyme protein, a method for producing the enzyme and a method for producing alcohol, particularly (2R,3R)-2,3-butanediol, by using the enzyme.
(R)-2,3-butanediol dehydrogenase is an enzyme which plays important roles in fermentation production of (2R,3R)-2,3-butanediol with microorganisms using glucose as raw material and in 2,3-butanediol metabolism in microorganisms. Further (2R,3R)-2,3-butanediol generated via the enzyme reaction is a useful compound as raw material for the synthesis of liquid crystal, pharmaceuticals, etc.
(R)-2,3-butanediol dehydrogenase is a dehydrogenase having the activity of selectively oxidizing the hydroxyl group of 2,3-butanediol in (R) configuration and also has the activity of oxidizing the hydroxyl group of meso-2,3-butanediol in (R) configuration as well as that of (2R,3R)-2,3-butanediol in (R) configuration.
Previously, regarding the enzyme having the activity of 2,3-butanediol dehydrogenation, it has been reported dehydrogenase activity toward (2R,3R)-2,3-butanediol is contained, for example, in the microorganisms listed below, based on studies concerning biosynthesis and metabolism of 2,3-butanediol (Arch. Microbiol., 116:197-203, 1978; J. Ferment. Technol., 61:467-471, 1983; J. Ferment. Technol., 62:551-559, 1984). However a variety of natures such as stereoselectivity and specific activity of 2,3-butanediol dehydrogenase was unclear in such previous studies because assays for the activity were conducted by using only cell-free extract and thus various enzymes coexisted:
Aeromonas hydrophila; 
Bacillus cereus IAM 1072;
Bacillus coagulans ATCC 8038;
Micrococcus lysodeikticus IAM 1056;
Micrococcus luteus IAM 1097;
Micrococcus roseus IAM 1295;
Pseudomonas saccharophila IAM 1504;
Sarcina lutea IAM 1099;
Staphylococcus aureus.
With respect to enzymes highly purified and having a variety of natures clarified, the following enzymes have been shown to have the activity of 2,3-butanediol dehydrogenase. However, these contain only the activity of catalyzing DL-form and there is no report on the stereoselectivity. Furthermore, the activities of the 2,3-butanediol dehydrogenases with the exception of Pichia ofunaensis are comparable to or lower than the activity of glycerol dehydrogenase and thus the specific activities are generally lower.
Glycerol dehydrogenase derived from Achromobacter liquidum (Achromobacter liquidum KY 3047) (Examined Published Japanese Patent Application No. (JP-B) Sho 58-40467);
Glycerol dehydrogenase derived from Bacillus sp. (Bacillus sp. G-1) (JP-B Hei 03-72272);
Glycerol dehydrogenase derived from Bacillus stearothermophilus (Biochim. Biophys. Acta., 994:270-279, 1989);
Glycerol dehydrogenase derived from Citrobacter freundii (Citrobacter freundii DSM 30040) (J. Bacteriol., 177:4392-4401, 1995);
Glycerol dehydrogenase derived from Erwinia aroideae (Erwinia aroideae IFO 3830) (Chem. Pharm. Bull., 26:716-721, 1978);
Glycerol dehydrogenase derived from Geotrichum candidum (Geotrichum candidum IFO 4597) (JP-B Hei 01-27715);
Dihydroxyacetone reductase derived from Pichia ofunaensis (Pichia ofunaensis AKU 4328) (J. Biosci. Bioeng., 88:148-152, 1999);
Glycerol dehydrogenase derived from Schizosaccharomyces pombe (J. Gen. Microbiol., 131:1581-1588, 1985).
A known enzyme highly purified and having a clarified high selectivity to (2R,3R) isomer of 2,3-butanediol is glycerol dehydrogenase produced by Escherichia coli (Escherichia coli W-1485) (J. Biol. Chem., 259:2124-2129, 1984). Because Vmax of this enzyme toward (2R,3R)-2,3-butanediol is 28.0 U/mg protein and Vmax toward racemic body is 21.2 U/mg protein, the enzyme is suggested to exhibit the stereoselectivity to (2R,3R) isomer. Here, 1 U of the enzyme is defined as an enzyme activity of reducing 1 xcexcmol oxidized nicotinamide adenine dinucleotide (hereinafter abbreviated to NAD+) into reduced nicotinamide adenine dinucleotide (hereinafter abbreviated to NADH) for one minute in the presence of (2R,3R)-2,3-butanediol as a substrate.
Further it has been reported that (R)-2,3-butanediol dehydrogenase derived from Saccharomyces cerevisiae produces (2R,3R)-2,3-butanediol from 2,3-butanedione (Arch. Microbiol., 154:267-273, 1990), but the dehydrogenase activity to DL-2,3-butanediol is about 20.3 U/mg protein; all of the above exhibit merely low specific activities.
In addition, the gene encoding 2,3-butanediol dehydrogenase participating in the metabolism of 2,3-butanediol has been cloned from Pseudomonas putida and expressed in E. coli (FEMS Microbiol. Lett., 124(2): 141-150, 1994), but the stereoselectivity has not yet been reported. Further genomic analysis has identified a gene from Pseudomonas aeruginosa, which has high homology to the 2,3-butanediol dehydrogenase gene derived from Pseudomonas putida. However this gene has not yet been expressed recombinantly and thus neither enzyme activity nor stereoselectivity has been verified.
The followings are industrially important challenges; the discovery of (R)-2,3-butanediol dehydrogenase that is useful for producing optically active alcohols such as (2R,3R)-2,3-butanediol, high stereoselectivity and high specific activity; particularly, the isolation of gene encoding the enzyme and preparation of transformants capable of expressing the enzyme to make it possible to conveniently produce the enzyme on a large scale.
An objective of the present invention is to provide (R)-2,3-butanediol dehydrogenase that can use NAD+ as a coenzyme. Another objective of the present invention is to provide (R)-2,3-butanediol dehydrogenase capable of giving products of high optical purity in high yield when it is utilized in an enzymatic production process of optically active (2R,3R)-2,3-butanediol using 2,3-butanedione as a substrate.
Yet another objective of the present invention is to isolate a polynucleotide encoding (R)-2,3-butanediol dehydrogenase having desired properties and to obtain a recombinant thereof. In addition, still another objective is to provide a method for enzymatically producing optically active (2R,3R)-2,3-butanediol by using the novel (R)-2,3-butanediol dehydrogenase.
The present inventors have studied a group of enzymes participating in glycerol metabolism in Pichia angusta (previous name: Hansenula polymorpha) (Agri. Biol. Chem., 51:2401-2407, 1987). There are two glycerol metabolism pathways, namely phosphorylation pathway and oxidation pathway, in this fungal strain; thus it has been clarified that the strain has both glycerol dehydrogenase I (GDH-I) catalyzing reduction reaction using NADH and dibydroxyacetone as substrates at pH 6.0 as well as glycerol dehydrogenase II (GDH-II) catalyzing oxidation reaction using NAD+ and glycerol as substrates at pH 9.0.
One of these two types of enzymes, GDH-I, was purified to a single band in electrophoresis and a variety of natures thereof have been clarified. The result showed that GDH-I is a novel (R)-2,3-butanediol dehydrogenase having the high activity as well as high selectivity to the hydroxyl group of 2,3-butanediol in (R) configuration.
Further, the present inventor isolated a polynucleotide encoding this enzyme and prepared recombinant bacteria overexpressing this enzyme, thereby completing the present invention. Specifically the present invention relates to the following (R)-2,3-butanediol dehydrogenase, a polynucleotide encoding this enzyme, a method for producing this enzyme and uses thereof.
[1] An (R)-2,3-butanediol dehydrogenase having the following physicochemical properties (1) to (3):
(1) Action
The dehydrogenase produces (R)-acetoin by acting on (2R,3R)-2,3-butanediol using nicotinamide adenine dinucleotide as a coenzyme. The dehydrogenase produces (2R,3R)-2,3-butanediol by reducing 2,3-butanedione using reduced form of nicotinamide adenine dinucleotide as a coenzyme;
(2) Substrate specificity
The dehydrogenase uses nicotinamide adenine dinucleotide as a coenzyme in oxidation reaction. The dehydrogenase uses reduced form of nicotinamide adenine dinucleotide as a coenzyme in reduction reaction. In addition, the dehydrogenase preferentially oxidizes a hydroxyl group of 2,3-butanediol in (R) configuration; and
(3) Specific activity:
The dehydrogenase has 100 U or higher of (R)-2,3-butanediol dehydrogenase activity per 1 mg of the dehydrogenase when purified.
[2] The (R)-2,3-butanediol dehydrogenase of [1], wherein the dehydrogenase further has the following physicochemical properties (4) and (5):
(4) Optimal pH
Optimal pH for glycerol oxidation reaction is 10; and
(5) Molecular weight
Molecular weight of a subunit of the dehydrogenase is 36,000 when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular weight of the dehydrogenase is 76,000 when determined by gel filtration.
[3] The (R)-2,3-butanediol dehydrogenase of [1], wherein the dehydrogenase is produced by a microorganism belonging to the genus Pichia. 
[4] The (R)-2,3-butanediol dehydrogenase of [3], wherein the microorganism is Pichia angusta. 
[5] A polynucleotide of (a) to (d) below:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1;
(b) a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
(c) a polynucleotide encoding a polypeptide that comprises an amino acid sequence comprising the amino acid sequence of SEQ ID NO:2 in which one or more amino acids are substituted, deleted, inserted, and/or added and that is functionally equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO:2; and
(d) a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 and that encodes a polypeptide functionally equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
[6] The polynucleotide of [5], wherein the polynucleotide comprises a nucleotide sequence having 70% or higher percent identity to the nucleotide sequence of SEQ ID NO:1.
[7] The polynucleotide of [5], wherein the polynucleotide encodes an amino acid sequence having 70% or higher percent identity to the amino acid sequence of SEQ ID NO:2.
[8] A polypeptide encoded by the polynucleotide of [5].
[9] The polypeptide of [8], wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.
[10] A vector comprising the polynucleotide of [5].
[11] A transformant comprising the polynucleotide of [5] or the vector of [10].
[12] A method for producing the polypeptide of [9], the method comprising the steps of:
culturing the transformant of [11] and
recovering an expression product.
[13] A method for producing the dehydrogenase of [1] or the polypeptide of [8], the method comprising culturing a microorganism that belongs to the genus Pichia and that produces the dehydrogenase of [1] or the polypeptide of [8].
[14] The method of [13], wherein the microorganism is Pichia angusta. 
[15] A method for producing an alcohol, the method comprising the steps of:
reacting a substance having (R)-2,3-butanediol dehydrogenase activity to a ketone in the presence of reduced form of nicotinamide adenine dinucleotide to generate the alcohol, wherein the substance is selected from the group consisting of the (R)-2,3-butanediol dehydrogenase of [1], the polypeptide of [8], a microorganism producing any one of them, and a processed product thereof, and
recovering the generated alcohol.
[16] The method of [15], wherein the microorganism is the transformant of [11].
[17] The method of [15], wherein the ketone is 2,3-butanedione and the alcohol is (2R,3R)-2,3-butanediol.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.